Objective: The purpose of this lab is to grow up colonies and eventually test which clones worked.
Analysis: Colonies were obtained from the master plate and grown in a tube with LB media + kanamycin for 19 hours. More bacteria were obtained and the bacteria pellets were prepared for the Mini-prep.
MiniPrep Kit 11/22/13 12:56 PM ~ 2:41 PM
Objective: The purpose of this lab is to extract DNA from the bacteria obtained from eight transformed tubes.
Figure 1. The concentration of final eluted DNA from cloned bacteria sample #1 after Mini-prep is measured via spectrophotometry. The concentration was 93.8 ng/µL. 260/280 was 1.86, while 260/230 was 1.98.
Figure 2. The concentration of final eluted DNA from cloned bacteria sample #2 after Mini-prep is measured via spectrophotometry. The concentration was 71.3 ng/µL. 260/280 was 1.85, while 260/230 was 1.90.
Figure 3. The concentration of final eluted DNA from cloned bacteria sample #3 after Mini-prep is measured via spectrophotometry. The concentration was 15.8 ng/µL. 260/280 was 1.72, while 260/230 was 1.50.
Figure 4. The concentration of final eluted DNA from cloned bacteria sample #4 after Mini-prep is measured via spectrophotometry. The concentration was 75.9 ng/µL. 260/280 was 1.85, while 260/230 was 1.85.
Figure 5. The concentration of final eluted DNA from cloned bacteria sample #5 after Mini-prep is measured via spectrophotometry. The concentration was 23.0 ng/µL. 260/280 was 1.82, while 260/230 was 1.59.
Figure 6. The concentration of final eluted DNA from cloned bacteria sample #6 after Mini-prep is measured via spectrophotometry. The concentration was 88.9 ng/µL. 260/280 was 1.84, while 260/230 was 1.96.
Figure 7. The concentration of final eluted DNA from cloned bacteria sample #7 after Mini-prep is measured via spectrophotometry. The concentration was 23.8 ng/µL. 260/280 was 1.82, while 260/230 was 1.73.
Figure 8. The concentration of final eluted DNA from cloned bacteria sample #8 after Mini-prep is measured via spectrophotometry. The concentration was 28.6 ng/µL. 260/280 was 2.02, while 260/230 was 1.77.
Analysis: Most of the samples had the concentration over 20 ng/µL except the sample #3. Therefore, all seven samples are going to be used to determine potential positive clones by submitting DNA to DNA sequencing facility.
Submitting DNA to DNA sequencing Facility 11/22/13 3:45 PM ~ 5:00 PM
Objective: The purpose of this experiment is to determine the potential positive clones.
Great job. -UM
Week 11 & 12
Cloning - Second Attempt
Cohesive end generation 11/04/2013 9:10 AM ~ 12:41 PM
Objective: The purpose of this lab is to create cohesive end generations on PCR inserts and accepting vector to prepare the samples for annealing and transformation.
Tube A: Mixed 2 µL of T4-treated Accepting Vector with 4µL of T4-treated Insert
Tube B: Mixed 1 µL of T4-treated Accepting Vector with 9µL of T4-treated Insert
Tube C: Mixed 2 µL of T4-treated Accepting Vector with 5µL of T4-treated Insert
Figure 1. Left to Right; LB-agar plate with 50 µg/ml kanamycin and 5% sucrose + Tube C, LB-agar plate with 50 µg/ml kanamycin and 5% sucrose + Tube B, LB-agar plate with 50 µg/ml kanamycin and 5% sucrose + Tube A
Analysis: Since BL21 competent cells were used instead of the DH5 alpha cells, it might have cause the growth of other bacteria or the fungi. Therefore, the DH5 alpha competent cells will be used in the next cloning attempt.
Cloning - Third Attempt
Cohesive end generation & Annealing and Transformation 11/07/2013 2:47 PM ~ 06:00 PM
Objective: The purpose of this lab is to create cohesive end generations on PCR inserts and accepting vector to prepare the samples for annealing and transformation.
Tube A: Mixed 2 µL of T4-treated Accepting Vector with 4µL of T4-treated Insert
Tube B: Mixed 1 µL of T4-treated Accepting Vector with 9µL of T4-treated Insert
Figure 1. Left to Right; LB-agar plate with 50 µg/ml kanamycin and 5% sucrose + 1:9 Tube, LB-agar plate with 50 µg/ml kanamycin and 5% sucrose + 2:4 Tube
Analysis: Using DH5 alpha cells were used instead of the BL21 competent cells, colonies were grew after the 48 hours of incubation. Therefore, eight colonies will be selected for the master plate.
Master Plate 3:30 ~ 4:45 PM 11/14/13
Objective: The purpose of this lab is to grow up colonies and eventually test which clones worked.
Figure 1. Eight colonies obtained from the two transformed bacteria plates were planted on the master plate with LB agar and the kanamycin.
Figure 2. Selected colonies were labeled with numbers and placed into the labeled tube accordingly for the more growth. Each tube contains transformed bacteria (#1 ~ #8), LB agar, and the kanamycin.
Analysis: Master plate was made to distinguish the eight colonies from the other colonies and incubated in the 37 degrees Celsius incubator, and eight tubes were incubated for 20 hours to grow more bacteria for the mini-prep.
MiniPrep Kit 11/15/13 2:39 PM ~ 4:15 PM
Objective: The purpose of this lab is to extract DNA from the bacteria obtained from eight transformed tubes.
Figure 1. The concentration of final eluted DNA from cloned bacteria sample #1 after Mini-prep is measured via spectrophotometry. The concentration was 12.2 ng/µL. 260/280 was 1.59, while 260/230 was 0.66.
Figure 2. The concentration of final eluted DNA from cloned bacteria sample #2 after Mini-prep is measured via spectrophotometry. The concentration was 6.5 ng/µL. 260/280 was 1.53, while 260/230 was 0.41.
Figure 3. The concentration of final eluted DNA from cloned bacteria sample #3 after Mini-prep is measured via spectrophotometry. The concentration was 12.4 ng/µL. 260/280 was 1.68, while 260/230 was 1.17.
Figure 4. The concentration of final eluted DNA from cloned bacteria sample #4 after Mini-prep is measured via spectrophotometry. The concentration was 19.3 ng/µL. 260/280 was 1.81, while 260/230 was 1.26.
Figure 5. The concentration of final eluted DNA from cloned bacteria sample #5 after Mini-prep is measured via spectrophotometry. The concentration was 11.4 ng/µL. 260/280 was 1.75, while 260/230 was 1.52.
Figure 6. The concentration of final eluted DNA from cloned bacteria sample # 6 after Mini-prep is measured via spectrophotometry. The concentration was 32.7 ng/µL. 260/280 was 1.66, while 260/230 was 0.69.
Figure 7. The concentration of final eluted DNA from cloned bacteria sample #7 after Mini-prep is measured via spectrophotometry. The concentration was 8.3 ng/µL. 260/280 was 1.34, while 260/230 was 0.56.
Figure 8. The concentration of final eluted DNA from cloned bacteria sample #8 after Mini-prep is measured via spectrophotometry. The concentration was 7.5 ng/µL. 260/280 was 1.65, while 260/230 was 1.03.
Analysis: The concentrations of final eluted DNA from cloned bacteria samples after mini-prep were too low which represent that the DNA are not in the tested samples. It was confirmed that the wash solution did not contain ethanol which might have caused the loss of DNA during the mini-prep. Therefore, mini-prep should be done again using the wash solution + ethanol.
Good work. Your concentrations for pNIC were great. Hope your transformation works this time! Could use more analysis on virtual work. -UM
Week 9 & 10
Transformation of competent cells for plasmid prep of pNIC-Bsa4 10/24/13 3:43 ~ 4:30 PM
Objective: The purpose of this lab is to transform bacteria using E.coli DH5α and pNIC-Bsa4 to make more plasmid DNA.
Analysis: Same protocol (past transformation trial) was used. Instead of using 80mL of LB and 0.9 µL of kanamycin, 80mL of LB and 80 µL of kanamycin was used. Two flasks were made (each flask had 80mL of LB, 80 µL of kanamycin and one colony from bacteria plate). Jesus' bacteria plate was used to grow pNIC (approved by Dr.B).
Midi Prep using HiSpeed Kit 10/25/13 11:40 AM ~ 1:30 PM
Objective: The purpose of this lab is to extract DNA from newly transformed bacterial cells.
Figure 1. The concentration of Final eluted pNIC-Bsa4 from newly transformed bacteria cells is measured via spectrophotometry. The concentration was 112.8 ng/µL. 260/280 was 1.87. 260/280 was 2.67.
Analysis: Since the concentration obtained from PCR clean-up (trial 1 ~ trial 4) for the accepting vector, the bacteria transformation was re-done using Jesus' bacteria colonies. It was confirmed that 80 µL of kanamycin should have used instead of 0.9 µL of kanamycin. After growing a new pNIC-Bsa4 culture using 80 mL of LB, 80 µL of kanamycin and a colony from Jesus' plate, Midi-prep was done using combined bacteria pallets which gave the concentration of 112.8 ng/µL. To increase the concentration, 0.5 mL of Buffer TE was used instead of 1 mL of Buffer TE. The concentration of 112.8 ng/µL was high enough to proceed to the cloning steps. 260/280 value - which was 1.87 - showed that the sample was pure. Alos, 260/230 - which was 2.67 - showed that the sample was not contaminated.
pNIC-Bsa4 Cloning
Objective: The purpose of this lab is to transfer pNIC-Bsa4 into a protein expression vector.
PCR Clean-Up for newly created accepting vectors after Midi Prep 10/25/13 5:00 ~ 7:00 PM
Figure 1. The concentration of final eluted pNIC accepting vectors from PCR clean-up is measured via spectrophotometry. The concentration was 42.0 ng/µL. 260/280 was 1.97, while 260/230 was 4.67.
Analysis: The concentration of the accepting vectors was 42 ng/µL. Since the solution was spilled during the lab, it might have affected the 260/230 value which shows the contamination. However, since the graph shows a clear peak at 260 wavelength, we could use the accepting vectors to clone.
Cohesive End Generation steps 10/28/13 2:00 ~ 4:07 PM
Objective: The purpose of this lab is to create cohesive end generations on PCR inserts and accepting vector to prepare the samples for annealing and transformation.
Figure1. First plate contains LB + Kan + Suc + 2 µL of accepting vector + 4 µL of PCR inserts. Second plate contains LB + Kan + Suc + 1µL of accepting vector + 9 µL of PCR inserts.
Analysis: After incubating both plates at 37 degrees Celsius, it was confirmed that both bacteria did not grow on neither plates. Low concentrations of both PCR products and accepting vector possibly inhibit the bacteria grow. Therefore, better concentrations should be obtained from PCR^2 products and the accepting vector.
Objective: The purpose of this lab is to create the homology model using Xlaunch, Xming, Putty and WinSCP programs. PCR^2 and PCR clean-up 10/29/13 4:00 ~ 5:30 PM
Objective: The purpose of this lab is to confirm that the PCR worked correctly and the oligo mix was made correctly. By adding forward and reverse primers into the secondary PCR solution, full length DNA created from primary PCR was amplified.
Figure 1. The concentration of cleaned PCR^2 product is measured via spectrophotometry. The concentration was 176.0 ng/µL. 260/280 was 1.86. 260/230 was 2.26.
Analysis: PCR^2 was re-conducted using the secondary PCR product to get higher concentration which will give better result for the cohesive end generation product.
PCR Clean-Up for newly created accepting vectors after Midi Prep 10/29/13 5:00 ~ 7:00 PM
Figure 1. The concentration of final eluted pNIC accepting vectors from PCR clean-up is measured via spectrophotometry. The concentration was 372.8 ng/µL. 260/280 was 1.86, while 260/230 was 2.33.
Analysis: The concentration of the accepting vectors was 372.8 ng/µL. Since four pNIC accepting vector solutions were cleaned up using PCR clean up kit, the better concentration was obtained which will give better result for the cohesive end generation product.
Good work. Could have more data. Try the pNIC again, concentrations are low. -UM
Week 7 & 8
pNIC-Bsa4 cloning
Preparation of pNIC-Bsa4 as Accepting Vector and PCR clean-up
Objective: The purpose of this experiment is to transfer Gene of Interest into a protein expression vector.
Trial1: 10/16/2013 1:13 PM ~ 4:52 PM
Figure 1: The concentration of DNA extracted from incubated pNIC-Bsa4 accepting vector was measured via spectrophotometry. The concentration was 5.6 ng/µL. 260/280 was 1.41, while 260/230 was 0.65.
Trial 2: 10/17/2013 2:20 PM ~ 2:41 PM
Figure 2: The concentration of DNA extracted from incubated pNIC-Bsa4 accepting vector was measured via spectrophotometry. The concentration was 1.5 ng/µL. 260/280 was 1.31, while 260/230 was 0.91.
Trial 3: 10/17/2013 2:38 PM ~ 6:35 PM
Figure 3:
The concentration of DNA extracted from incubated pNIC-Bsa4 accepting vector was measured via spectrophotometry. The concentration was 2.5 ng/µL. 260/280 was 1.00, while 260/230 was 1.10.
Trial 4: 10/18/2013 4:38 PM ~ 8:00 PM
Figure 4:
The concentration of DNA extracted from incubated pNIC-Bsa4 accepting vector was measured via spectrophotometry. The concentration was 5.8 ng/µL. 260/280 was 2.11, while 260/230 was 0.41.
Analysis: For trial 1-3, ethanol was not added into a wash solution which might have caused the low concentration of the samples. Therefore, trial 4 was conducted by using wash solution+ethanol which still resulting a very low concentration. Therefore, bacteria transformation needs to be conducted again to obtain a better result.
Objective: The purpose of this lab is to extract DNA from transformed bacteria cells using plasmid prep of pNIC-Bsa4.
Figure1. The concentration of extracted DNA from transformed bacteria cells is measured vis spectrophotometry. The concentration was 31.1 ng/µL. 260/280 was 1.90, while 260/230 was -2.64.
Analysis: DNA from the transformed bacteria cells was extracted using HiSpeed Plasmid Midi kit. After the cells were lysed using buffered in the kit, DNA was separated from other unnecessary components (cell membrane and proteins). The concentration of extracted DNA from the transformed bacteria cells was measure using nano drop which was 31.1 ng/µL. 260/280 value - which was 1.90 - showed that the sample used was pure. However, 260/230 value - -2.64 - represented that the DNA sample was contaminated.
Great work! I am liking the progress that I'm seeing. Keep up the hard work! - Michael T.
Week 5 & 6
Secondary PCR
Objective: The purpose of this lab is to confirm that the PCR worked correctly and the oligo mix was made correctly. By adding forward and reverse primers into the secondary PCR solution, full length DNA created from primary PCR was amplified.
Trial #1 - 09/20/13 1:00 ~ 5:30 PM
Figure 1. An image of gel electrophoresis for the secondary PCR. Lane 1 is empty. Lane 2 is showing 100 bp ladder. Lane 3 contains primary PCR, both Forward and Reverse primers, Q5 hotstart polymerase, 5X rnx buffer, 2mM dNTPs and autoclaved nano pure water. However, sample was not shown.
Analysis: The sample for secondary PCR was not shown in the gel.
Trial 2 - 09/26/13 2:15 ~ 7:30 PM
Figure 1. An image of gel electrophoresis for the secondary PCR - trial 2. Lane 1 is empty. Lane 2 contains 100bp ladder. Lane 3 contains primary PCR, both Forward and Reverse primers, Q5 hotstart polymerase, 5X rnx buffer, 2mM dNTPs and autoclaved nano pure water. However, sample was not shown.
Analysis: Nothing showed on the gel. Possible errors were contamination of the sample and wrong annealing temperature for DNA. Gel was heated for 4 hours which possibly made all the samples including DNA ladder and samples to ran off.
Trial 3 - 09/27/13 3:30 ~ 7:28 PM
Figure 1. An image of gel electrophoresis for the secondary PCR - trial 3. Lane 1 is empty. Lane 2 contains 1kb Ladder. Lane 3 is empty. lane 4 contains 100bp ladder. Lane 5 contains primary PCR, both Forward and Reverse primers, Q5 hotstart polymerase, 5X rnx buffer, 2mM dNTPs and autoclaved nano pure water.
Figure 2. 100 bp DNA Ladder visualized by ethidium bromide staining on a 1.3% TAE agarose gel. Mass values are fore 0.5 µg/lane. Image of the ladder and the description was obtained from New England BioLabs Inc. website.
Figure 3. 1kb DNA Ladder visualized by ethidium bromide staining on a 0.8% TAE agarose gel. Mass values are fore 0.5 µg/lane. Image of the ladder and the description was obtained from New England BioLabs Inc. website.
Analysis: The band contains the sample obtained from secondary PCR was clearly shown. Both 1kb and 100bp was used to compare the size of DNA. The band is located between approximately 700 and 800 base pairs according to 100 bp ladder. Therefore, secondary PCR was successful since the size of nucleotide of the sample used for this lab was 741 nucleotides.
PCR^2 - 09/26/2013 04:46 ~ 08:21 PM
Figure 1. An image of gel electrophoresis for PCR^2. Lane 1 contains 1kb DNA Ladder. From Lane 2-5, each contains 50 µL containing sample obtained from secondary PCR, 5X rxn buffer, 2mM dNTPs, Q5 hotstart polymerase, both Forward and Reverse Primers and autoclaved nanopure water.
Figure 2. 1kb DNA Ladder visualized by ethidium bromide staining on a 0.8% TAE agarose gel. Mass values are fore 0.5 µg/lane. Image of the ladder and the description was obtained from New England BioLabs Inc. website.
Analysis: Since the solution was approximately 220 µL which was oversized to run the PCR, solution was divided into four PCR tubes. Therefore, all four bands appear at the same location with the same intensity. Ironically, even though the secondary PCR did not clearly show the result in previous step, PCR^2 successfully worked by showing all four bands located at approximately 700~800 bases which was close to the actual DNA size, 731 nucleotides. From the result obtained from PCR^2, I could conclude that the gel was heated too long which probably made all samples to ran off.
LB agar plate or LB media - 09/26/13 3:48 ~ 07:26 PM
Objective: The purpose of this lab is to make 160mL of LB media and four agar plates using LB and Kanamysin for future experiment.
Analysis: Four LB agar plates were made using 80mL of LB and 80µL of Kanamycin. Kanamycin stock was used instead of Ampicillin stock, since pNIC-Bsa4 is Kanamycin resistance bacteria. On the other hand, antibiotic was not added into LB media.
PyMol Refresher - 09/29/13 3:30 ~ 6:45 PM
Objective: The purpose of this lab is to examine three dimensional structure of different enzymes.
Analysis: By comparing polar contacts, shape of inhibitor residues and binding mode at the active site, one could use these factors to determine similarities/differences of different enzymes. Moreover, one could figure out that each NAP residues are located at each chain from 3HBB enzymes, since the3HBB enzyme was composed of four different chains. Human and bacterial proteins were compared by aligning two proteins together using PyMol. By using BLAST, similarities/differences were determined.Different enzymes including 3HBB, 1U72, 3CL9 and 2H2Q are analyzed to find substrates, inhibitors and cofactors of different enzymes. Inhibitor MTX for human protein and TMQ for bacterial protein were similar, however, MTX binds better to the active site and has more residues to interact with.
PCR CleanUp & Nanodrop 10/01/13 2:18 ~ 3:12 PM
Objective: The purpose of this lab is to determine concentration of DNA by measuring absorbance by using spectrophotometry.
Figure 1. The concentration of cleaned PCR^2 product is measured via spectrophotometry. The concentration was 3.5 ng/µL. 260/280 was 1.81. 260/230 was 0.93.
Analysis: The absorbances at two different wavelengths were taken to estimate the purity of DNA eluted from PCR^2 samples. The concentration was 3.6 ng/µL which was incorrect, since the value for concentration was too low. Also, 260/280 was 1.81 which shows that the DNA sample used in this lab was pure since the ideal 260/280 is 1.8. 260/230 value - which shows the presence of other contaminants - was 0.93 which represents that the DNA sample was contaminated. DNA samples could possibly ran off with wash solutions which caused wrong concentration.
Transformation of competent cells for plasmid prep of pNIC-Bsa4 10/01/13 ~ 10/03/13
Objective: The purpose of this lab is to transform bacteria using E.coli DH5α and pNIC-Bsa4 to make more plasmid DNA.
Day 1 : 10/01/13 03:12 PM ~ 05:05 PM Day 2: 10/02/13 6:00 ~ 6:34 PM Day 3: 10/03/13 11:22 ~ 12:09 PM
Figure1. (From left to right) pallet obtained from the solution containing E.coli DHα5 (10µL) + pNIC-Bsa4 + LB + Kanamycin,
obtained from the solution containing E.coli DHα5 (50µL) + pNIC-Bsa4 + LB + Kanamysin, obtained from the solution containing E.coli DHα5 (50µL) + pNIC-Bsa4 + LB + Kanamycin, obtained from the solution containing E.coli DHα5 (10µL) + pNIC-Bsa4 + LB + Kanamycin
Analysis: On the first day, bacteria was incubated in LB agar plate with Kanamycin for approximately 25 hours. The next day, one bacteria colony was collected to place into LB media and Kanamycin was added which was finally placed in 37 degree shaker at fast rpm 250 rpm for 16 hours. On the last day, four flasks containing bacteria + LB media + Kanamycin was span down in 4 degree centrifuge at 6000 x g for 15 mins, and then only bacteria pallet was kept in -20 degree freezer.
Week 3 & 4
Kelly - good work. Include a ladder image. Dr. B 092713
PCR Primer Design Tails for pNIC-Bsa4 Cloning 09/12/2013 4:56 ~ 6:24 PM
Objective: The purpose of this lab was to design both Forward and Reverse primers for PCR amplifying the CDS of Alpha Carbonic Anhydrase and synthesizing ends of gene for the insertion into the pNIC-Bsa4. Forward Primer
Melt Temp
0mM Mg 2+: 58.1 C
1.5mM Mg 2+: 65.8 C
2mM Mg 2+: 66.4 C
4mM Mg 2+: 67.5 C
6mM Mg 2+: 68.1 C
Reverse Primer
5' TCT GCG AAA ACC CGT TAA CAG TAA AGG TGG ATA 3'
30 bp
GC Content: 42.4 %
Melt Temp
0mM Mg 2+: 62.6 C
0.5mM Mg 2+: 70.0 C
2mM Mg 2+: 70.5 C
4mM Mg 2+: 71.5 C
6mM Mg 2+: 72.0 C
Virtual Plasmid
NEB Cutter - Gel view
Figure 1. Gel view of pNIC-Bsa4 cut with Bsa I
Gel view of DNA sequence inserted in pNIC-Bsa4 cut with Bsa I was not found.
Analysis: LIC Cloning was done using the coding sequence for Alpha Carbonate Dehydratase with both upstream and downstream primers. The sequence was inserted to pNIC-Bsa4 and was cut with the restriction enzyme BsaI. However, get view of DNA sequence inserted into pNIC-Bsa4 which was cut with BsaI was not found.
Re-do PCR 09/13/2013 2:45 ~ 8:00 PM
objective: The purpose of this lab is to amplify the coding sequence in the pGBR-22 using Forward and Reverse Primers and using four different samples with different concentration of DNA plasmid.
Figrue 1. Picture of gel electrophoresis obtained from PCR trial 2 (re-done). Lane 1 shows 100bp marker (DNA Ladder). Lane 2 shows sample A with the concentration of 0.3 ng/µL pGBR-22 which obtained from 1:1000 template dilution. Lane 3 shows sample B with the concentration of 3 ng/µL pGBR-22 which obtained from 1:1000 template dilution. Lane 4 shows sample C with the concentration of 30 ng/µL pGBR-22 which obtained from 1:100 template dilution. Lane 5 shows the sample D with no plasmid included.
Figure 2. 100 bp DNA Ladder visualized by ethidium bromide staining on a 1.3% TAE agarose gel. Mass values are fore 0.5 µg/lane. Image of the ladder and the description was obtained from New England BioLabs Inc. website.
Analysis: pGBR-22 plasmid was diluted to either 1:1000 or 1:100 and the diluted plasmid was sued to create four different samples with different amount of plasmid. Lane 5, with no plasmid, did not have a band shown, since the sample did not have any DNA plasmid included. On the other hand, all other lanes clearly showed bands with different intensity. lane 4 had the highest intensity, meaning sample C had the most amount of DNA plasmid. However, it was hard to distinguish the different between land 2 and lane 3 due to the similar intensity which might have been resulted from dilution error. Moreover, the size of sample was approximately 1000 base pairs.
Further Analysis: According to NEBcutter, two cuts were approximately at 1000bp. These two cuts were similar to the lane 5 from figure 1 which indicates the site cut with PvuII enzyme.
Objective: The purpose of this lab was to digest pGBR-22 plasmid with different restriction enzymes including EcoRI, PvuII and both EcoRI + PvuII and visualize fragments using gel electrophoresis.
Figure 1. Picture of gel electrophoresis obtained from Restriction Enzyme Digest. Lane 1 contains 1kb DNA Ladder. Lane 2 contains uncut pGBR-22 plasmid with the concentration of 48.95 ng/µL. Lane 3 contains pGBR-22 digested with BsaI. Lane 4 contains pGBR-22 digested with PvuII. Lane 5 contains pGBR-22 digested with both BsaI and PvuII.
Figure 2. 1kb DNA Ladder visualized by ethidium bromide staining on a 0.8% TAE agarose gel. Mass values are fore 0.5 µg/lane. Image of the ladder and the description was obtained from New England BioLabs Inc. website.
Analysis:
pGBR-22 plasmid was digested with BsaI instead of EcoRI. Wrong enzyme still cut the plasmid. Lane 3 showed that plasmid digested with BsaI was at 4kb. Lane 4 showed that plasmid digested with PvuII was at 3kb and 1kb. Lane 5 showed plasmid digested with PvuII and BsaI was at 1.5kb and 1kb. Lane 5 only showed two abnds which indicates that incorrect enzyme might cut the same site where PvuII enzyme would cut.
PCR Primer Overlap
Primer Dilutions for Assembly Step09/12/2013 6:00 ~ 7:20 PM
Objective: The purpose for this lab was to make an oligo Mix consisting of all of the primers for the Primary PCR, Secondary PCR and the PCR^2.
Analysis: Oligo Mix was made for further experiment with the Primers obtained from IDT.
Analysis: Forward and Reverse primers for HpAlphaCA sequence were ordered and will be used for Secondary PCR.
I. Primary PCR 9/19/2013 3:00 ~ 6:30 PM
Figure 1. Picture of gel electrophoresis obtained from the primary PCR. Lane 1 is empty. Lane 2 contains 100 bp DNA ladder. Lane 3 contains primary PCR solution with oligo mix, dNTP, 5X rxn buffer, Q5 Polymerase and dH2O.
Figure 2. 100 bp DNA Ladder visualized by ethidium bromide staining on a 1.3% TAE agarose gel. Mass values are fore 0.5 µg/lane. Image of the ladder and the description was obtained from New England BioLabs Inc. website.
Analysis: Since oligo mix was added to the primary PCR solution, land 3 has a smear. However, even though a clear band did not occur, one could see a DNA was mostly gathered at 500 bp.
Primer Dilution 9/19/2013 4:00 ~4:30 PM
Objective: The purpose of this lab is to dilute primers ordered for cloning
Analysis: Each of HpAlphaCA forward and reverse primers was separately mixed with TE in order to make 100 µM of the stock solution to the original container. After the stock solution was made using TE and primer powder, working dilutions were made by adding 40 µL of forward and reverse primers separately into two different 1.7ml centrifuge tubes and adding 160 µL of autoclaved nanopure water into each of two tubes. Both stock solutions and working dilutions were stored in -20 C freezer for future experiment.
Week 1 & 2
PCR Primer Design for Primer Overlap Assembly PCR
Objective: The purpose of this lab is to design an Oligo primer set of forward and reverse for PCR using the gene obtained from pGBR-22.
Analysis: In this experiment, Oligo-primer for PCR for cloning was designed. Also, the gene sequence from NCBI was obtained from NCBI and the primer plate result from Helix system-CIT.
Quantifying DNA Using Nanodrop
Objective: The purpose of this lab is to measure the absorbance including 26/280 and 260/230 values and the concentration of pGBR-22 plasmid.
Figure 2. First result for pGBR-22 plasmid with the concentration of 162.2 ng/µL and the absorbance of 1.151.
Figure 3. Second result for pGBR-22 plasmid with the concentration of 164.8 ng/µL and the absorbance of 1.179.
Analysis: The graph shows that results obtained from Nanodrop was close to actual, since the actual concentration was 156.92 ng/µL and the experimental concentration was approximately 163.5 ng/µL. Also, we could conclude that there was very little contamination, since 260/280 and 260/230 were close to 1.8 and 2.1. Possible errors were contamination in samples and measurement errors.
Submitting DNA and DNA Sequencing Facility
Objective: The purpose for this lab is to add a primer to pGBR-22.
Analysis:
Analyzing DNA Sequence
Objective: The purpose for this lab is to determine the DNA sequence of pGBR-22 plasmid.
Analysis: DNA sequence of a plasmid was obtained from BLAST and sequence manipulation suite to get an entire sequence for pGBR-22 protein.
Master Plate 5:19PM 11/21/13 ~ 12:24 PM 11/22/13
Objective: The purpose of this lab is to grow up colonies and eventually test which clones worked.
Analysis: Colonies were obtained from the master plate and grown in a tube with LB media + kanamycin for 19 hours. More bacteria were obtained and the bacteria pellets were prepared for the Mini-prep.
MiniPrep Kit 11/22/13 12:56 PM ~ 2:41 PM
Objective: The purpose of this lab is to extract DNA from the bacteria obtained from eight transformed tubes.
Figure 1. The concentration of final eluted DNA from cloned bacteria sample #1 after Mini-prep is measured via spectrophotometry. The concentration was 93.8 ng/µL. 260/280 was 1.86, while 260/230 was 1.98.
Figure 2. The concentration of final eluted DNA from cloned bacteria sample #2 after Mini-prep is measured via spectrophotometry. The concentration was 71.3 ng/µL. 260/280 was 1.85, while 260/230 was 1.90.
Figure 3. The concentration of final eluted DNA from cloned bacteria sample #3 after Mini-prep is measured via spectrophotometry. The concentration was 15.8 ng/µL. 260/280 was 1.72, while 260/230 was 1.50.
Figure 4. The concentration of final eluted DNA from cloned bacteria sample #4 after Mini-prep is measured via spectrophotometry. The concentration was 75.9 ng/µL. 260/280 was 1.85, while 260/230 was 1.85.
Figure 5. The concentration of final eluted DNA from cloned bacteria sample #5 after Mini-prep is measured via spectrophotometry. The concentration was 23.0 ng/µL. 260/280 was 1.82, while 260/230 was 1.59.
Figure 6. The concentration of final eluted DNA from cloned bacteria sample #6 after Mini-prep is measured via spectrophotometry. The concentration was 88.9 ng/µL. 260/280 was 1.84, while 260/230 was 1.96.
Figure 7. The concentration of final eluted DNA from cloned bacteria sample #7 after Mini-prep is measured via spectrophotometry. The concentration was 23.8 ng/µL. 260/280 was 1.82, while 260/230 was 1.73.
Figure 8. The concentration of final eluted DNA from cloned bacteria sample #8 after Mini-prep is measured via spectrophotometry. The concentration was 28.6 ng/µL. 260/280 was 2.02, while 260/230 was 1.77.
Analysis: Most of the samples had the concentration over 20 ng/µL except the sample #3. Therefore, all seven samples are going to be used to determine potential positive clones by submitting DNA to DNA sequencing facility.
Submitting DNA to DNA sequencing Facility 11/22/13 3:45 PM ~ 5:00 PM
Objective: The purpose of this experiment is to determine the potential positive clones.
Great job. -UM
Week 11 & 12
Cloning - Second Attempt
Cohesive end generation 11/04/2013 9:10 AM ~ 12:41 PM
Objective: The purpose of this lab is to create cohesive end generations on PCR inserts and accepting vector to prepare the samples for annealing and transformation.
Tube A: Mixed 2 µL of T4-treated Accepting Vector with 4µL of T4-treated Insert
Tube B: Mixed 1 µL of T4-treated Accepting Vector with 9µL of T4-treated Insert
Tube C: Mixed 2 µL of T4-treated Accepting Vector with 5µL of T4-treated Insert
Figure 1. Left to Right; LB-agar plate with 50 µg/ml kanamycin and 5% sucrose + Tube C, LB-agar plate with 50 µg/ml kanamycin and 5% sucrose + Tube B, LB-agar plate with 50 µg/ml kanamycin and 5% sucrose + Tube A
Analysis: Since BL21 competent cells were used instead of the DH5 alpha cells, it might have cause the growth of other bacteria or the fungi. Therefore, the DH5 alpha competent cells will be used in the next cloning attempt.
Cloning - Third Attempt
Cohesive end generation & Annealing and Transformation 11/07/2013 2:47 PM ~ 06:00 PM
Objective: The purpose of this lab is to create cohesive end generations on PCR inserts and accepting vector to prepare the samples for annealing and transformation.
Tube A: Mixed 2 µL of T4-treated Accepting Vector with 4µL of T4-treated Insert
Tube B: Mixed 1 µL of T4-treated Accepting Vector with 9µL of T4-treated Insert
Figure 1. Left to Right; LB-agar plate with 50 µg/ml kanamycin and 5% sucrose + 1:9 Tube, LB-agar plate with 50 µg/ml kanamycin and 5% sucrose + 2:4 Tube
Analysis: Using DH5 alpha cells were used instead of the BL21 competent cells, colonies were grew after the 48 hours of incubation. Therefore, eight colonies will be selected for the master plate.
Master Plate 3:30 ~ 4:45 PM 11/14/13
Objective: The purpose of this lab is to grow up colonies and eventually test which clones worked.
Figure 1. Eight colonies obtained from the two transformed bacteria plates were planted on the master plate with LB agar and the kanamycin.
Figure 2. Selected colonies were labeled with numbers and placed into the labeled tube accordingly for the more growth. Each tube contains transformed bacteria (#1 ~ #8), LB agar, and the kanamycin.
Analysis: Master plate was made to distinguish the eight colonies from the other colonies and incubated in the 37 degrees Celsius incubator, and eight tubes were incubated for 20 hours to grow more bacteria for the mini-prep.
MiniPrep Kit 11/15/13 2:39 PM ~ 4:15 PM
Objective: The purpose of this lab is to extract DNA from the bacteria obtained from eight transformed tubes.
Figure 1. The concentration of final eluted DNA from cloned bacteria sample #1 after Mini-prep is measured via spectrophotometry. The concentration was 12.2 ng/µL. 260/280 was 1.59, while 260/230 was 0.66.
Figure 2. The concentration of final eluted DNA from cloned bacteria sample #2 after Mini-prep is measured via spectrophotometry. The concentration was 6.5 ng/µL. 260/280 was 1.53, while 260/230 was 0.41.
Figure 3. The concentration of final eluted DNA from cloned bacteria sample #3 after Mini-prep is measured via spectrophotometry. The concentration was 12.4 ng/µL. 260/280 was 1.68, while 260/230 was 1.17.
Figure 4. The concentration of final eluted DNA from cloned bacteria sample #4 after Mini-prep is measured via spectrophotometry. The concentration was 19.3 ng/µL. 260/280 was 1.81, while 260/230 was 1.26.
Figure 5. The concentration of final eluted DNA from cloned bacteria sample #5 after Mini-prep is measured via spectrophotometry. The concentration was 11.4 ng/µL. 260/280 was 1.75, while 260/230 was 1.52.
Figure 6. The concentration of final eluted DNA from cloned bacteria sample # 6 after Mini-prep is measured via spectrophotometry. The concentration was 32.7 ng/µL. 260/280 was 1.66, while 260/230 was 0.69.
Figure 7. The concentration of final eluted DNA from cloned bacteria sample #7 after Mini-prep is measured via spectrophotometry. The concentration was 8.3 ng/µL. 260/280 was 1.34, while 260/230 was 0.56.
Figure 8. The concentration of final eluted DNA from cloned bacteria sample #8 after Mini-prep is measured via spectrophotometry. The concentration was 7.5 ng/µL. 260/280 was 1.65, while 260/230 was 1.03.
Analysis: The concentrations of final eluted DNA from cloned bacteria samples after mini-prep were too low which represent that the DNA are not in the tested samples. It was confirmed that the wash solution did not contain ethanol which might have caused the loss of DNA during the mini-prep. Therefore, mini-prep should be done again using the wash solution + ethanol.
Good work. Your concentrations for pNIC were great. Hope your transformation works this time! Could use more analysis on virtual work. -UM
Week 9 & 10
Transformation of competent cells for plasmid prep of pNIC-Bsa4 10/24/13 3:43 ~ 4:30 PM
Objective: The purpose of this lab is to transform bacteria using E.coli DH5α and pNIC-Bsa4 to make more plasmid DNA.
Analysis: Same protocol (past transformation trial) was used. Instead of using 80mL of LB and 0.9 µL of kanamycin, 80mL of LB and 80 µL of kanamycin was used. Two flasks were made (each flask had 80mL of LB, 80 µL of kanamycin and one colony from bacteria plate). Jesus' bacteria plate was used to grow pNIC (approved by Dr.B).
Midi Prep using HiSpeed Kit 10/25/13 11:40 AM ~ 1:30 PM
Objective: The purpose of this lab is to extract DNA from newly transformed bacterial cells.
Figure 1. The concentration of Final eluted pNIC-Bsa4 from newly transformed bacteria cells is measured via spectrophotometry. The concentration was 112.8 ng/µL. 260/280 was 1.87. 260/280 was 2.67.
Analysis: Since the concentration obtained from PCR clean-up (trial 1 ~ trial 4) for the accepting vector, the bacteria transformation was re-done using Jesus' bacteria colonies. It was confirmed that 80 µL of kanamycin should have used instead of 0.9 µL of kanamycin. After growing a new pNIC-Bsa4 culture using 80 mL of LB, 80 µL of kanamycin and a colony from Jesus' plate, Midi-prep was done using combined bacteria pallets which gave the concentration of 112.8 ng/µL. To increase the concentration, 0.5 mL of Buffer TE was used instead of 1 mL of Buffer TE. The concentration of 112.8 ng/µL was high enough to proceed to the cloning steps. 260/280 value - which was 1.87 - showed that the sample was pure. Alos, 260/230 - which was 2.67 - showed that the sample was not contaminated.
pNIC-Bsa4 Cloning
Objective: The purpose of this lab is to transfer pNIC-Bsa4 into a protein expression vector.
PCR Clean-Up for newly created accepting vectors after Midi Prep 10/25/13 5:00 ~ 7:00 PM
Figure 1. The concentration of final eluted pNIC accepting vectors from PCR clean-up is measured via spectrophotometry. The concentration was 42.0 ng/µL. 260/280 was 1.97, while 260/230 was 4.67.
Analysis: The concentration of the accepting vectors was 42 ng/µL. Since the solution was spilled during the lab, it might have affected the 260/230 value which shows the contamination. However, since the graph shows a clear peak at 260 wavelength, we could use the accepting vectors to clone.
Cohesive End Generation steps 10/28/13 2:00 ~ 4:07 PM
Objective: The purpose of this lab is to create cohesive end generations on PCR inserts and accepting vector to prepare the samples for annealing and transformation.
Figure1. First plate contains LB + Kan + Suc + 2 µL of accepting vector + 4 µL of PCR inserts. Second plate contains LB + Kan + Suc + 1µL of accepting vector + 9 µL of PCR inserts.
Analysis: After incubating both plates at 37 degrees Celsius, it was confirmed that both bacteria did not grow on neither plates. Low concentrations of both PCR products and accepting vector possibly inhibit the bacteria grow. Therefore, better concentrations should be obtained from PCR^2 products and the accepting vector.
Virtual Screening - Creating Homology Model 10/31/13 3:00 ~ 4:00 PM
Objective: The purpose of this lab is to create the homology model using Xlaunch, Xming, Putty and WinSCP programs.
PCR^2 and PCR clean-up 10/29/13 4:00 ~ 5:30 PM
Objective: The purpose of this lab is to confirm that the PCR worked correctly and the oligo mix was made correctly. By adding forward and reverse primers into the secondary PCR solution, full length DNA created from primary PCR was amplified.
Figure 1. The concentration of cleaned PCR^2 product is measured via spectrophotometry. The concentration was 176.0 ng/µL. 260/280 was 1.86. 260/230 was 2.26.
Analysis: PCR^2 was re-conducted using the secondary PCR product to get higher concentration which will give better result for the cohesive end generation product.
PCR Clean-Up for newly created accepting vectors after Midi Prep 10/29/13 5:00 ~ 7:00 PM
Figure 1. The concentration of final eluted pNIC accepting vectors from PCR clean-up is measured via spectrophotometry. The concentration was 372.8 ng/µL. 260/280 was 1.86, while 260/230 was 2.33.
Analysis: The concentration of the accepting vectors was 372.8 ng/µL. Since four pNIC accepting vector solutions were cleaned up using PCR clean up kit, the better concentration was obtained which will give better result for the cohesive end generation product.
Good work. Could have more data. Try the pNIC again, concentrations are low. -UM
Week 7 & 8
pNIC-Bsa4 cloning
Preparation of pNIC-Bsa4 as Accepting Vector and PCR clean-up
Objective: The purpose of this experiment is to transfer Gene of Interest into a protein expression vector.
Trial1: 10/16/2013 1:13 PM ~ 4:52 PM
Figure 1: The concentration of DNA extracted from incubated pNIC-Bsa4 accepting vector was measured via spectrophotometry. The concentration was 5.6 ng/µL. 260/280 was 1.41, while 260/230 was 0.65.
Trial 2: 10/17/2013 2:20 PM ~ 2:41 PM
Figure 2: The concentration of DNA extracted from incubated pNIC-Bsa4 accepting vector was measured via spectrophotometry. The concentration was 1.5 ng/µL. 260/280 was 1.31, while 260/230 was 0.91.
Trial 3: 10/17/2013 2:38 PM ~ 6:35 PM
Figure 3:
The concentration of DNA extracted from incubated pNIC-Bsa4 accepting vector was measured via spectrophotometry. The concentration was 2.5 ng/µL. 260/280 was 1.00, while 260/230 was 1.10.
Trial 4: 10/18/2013 4:38 PM ~ 8:00 PM
Figure 4:
The concentration of DNA extracted from incubated pNIC-Bsa4 accepting vector was measured via spectrophotometry. The concentration was 5.8 ng/µL. 260/280 was 2.11, while 260/230 was 0.41.
Analysis: For trial 1-3, ethanol was not added into a wash solution which might have caused the low concentration of the samples. Therefore, trial 4 was conducted by using wash solution+ethanol which still resulting a very low concentration. Therefore, bacteria transformation needs to be conducted again to obtain a better result.
Midi Prep Kit - HiSpeed 10/08/2013 2:22PM ~ 5:26PM
Objective: The purpose of this lab is to extract DNA from transformed bacteria cells using plasmid prep of pNIC-Bsa4.
Figure1. The concentration of extracted DNA from transformed bacteria cells is measured vis spectrophotometry. The concentration was 31.1 ng/µL. 260/280 was 1.90, while 260/230 was -2.64.
Analysis: DNA from the transformed bacteria cells was extracted using HiSpeed Plasmid Midi kit. After the cells were lysed using buffered in the kit, DNA was separated from other unnecessary components (cell membrane and proteins). The concentration of extracted DNA from the transformed bacteria cells was measure using nano drop which was 31.1 ng/µL. 260/280 value - which was 1.90 - showed that the sample used was pure. However, 260/230 value - -2.64 - represented that the DNA sample was contaminated.
Great work! I am liking the progress that I'm seeing. Keep up the hard work! - Michael T.
Week 5 & 6
Secondary PCR
Objective: The purpose of this lab is to confirm that the PCR worked correctly and the oligo mix was made correctly. By adding forward and reverse primers into the secondary PCR solution, full length DNA created from primary PCR was amplified.
Trial #1 - 09/20/13 1:00 ~ 5:30 PM
Figure 1. An image of gel electrophoresis for the secondary PCR. Lane 1 is empty. Lane 2 is showing 100 bp ladder. Lane 3 contains primary PCR, both Forward and Reverse primers, Q5 hotstart polymerase, 5X rnx buffer, 2mM dNTPs and autoclaved nano pure water. However, sample was not shown.
Analysis: The sample for secondary PCR was not shown in the gel.
Trial 2 - 09/26/13 2:15 ~ 7:30 PM
Figure 1. An image of gel electrophoresis for the secondary PCR - trial 2. Lane 1 is empty. Lane 2 contains 100bp ladder. Lane 3 contains primary PCR, both Forward and Reverse primers, Q5 hotstart polymerase, 5X rnx buffer, 2mM dNTPs and autoclaved nano pure water. However, sample was not shown.
Analysis: Nothing showed on the gel. Possible errors were contamination of the sample and wrong annealing temperature for DNA. Gel was heated for 4 hours which possibly made all the samples including DNA ladder and samples to ran off.
Trial 3 - 09/27/13 3:30 ~ 7:28 PM
Figure 1. An image of gel electrophoresis for the secondary PCR - trial 3. Lane 1 is empty. Lane 2 contains 1kb Ladder. Lane 3 is empty. lane 4 contains 100bp ladder. Lane 5 contains primary PCR, both Forward and Reverse primers, Q5 hotstart polymerase, 5X rnx buffer, 2mM dNTPs and autoclaved nano pure water.
Figure 2. 100 bp DNA Ladder visualized by ethidium bromide staining on a 1.3% TAE agarose gel. Mass values are fore 0.5 µg/lane. Image of the ladder and the description was obtained from New England BioLabs Inc. website.
Figure 3. 1kb DNA Ladder visualized by ethidium bromide staining on a 0.8% TAE agarose gel. Mass values are fore 0.5 µg/lane. Image of the ladder and the description was obtained from New England BioLabs Inc. website.
Analysis: The band contains the sample obtained from secondary PCR was clearly shown. Both 1kb and 100bp was used to compare the size of DNA. The band is located between approximately 700 and 800 base pairs according to 100 bp ladder. Therefore, secondary PCR was successful since the size of nucleotide of the sample used for this lab was 741 nucleotides.
PCR^2 - 09/26/2013 04:46 ~ 08:21 PM
Figure 1. An image of gel electrophoresis for PCR^2. Lane 1 contains 1kb DNA Ladder. From Lane 2-5, each contains 50 µL containing sample obtained from secondary PCR, 5X rxn buffer, 2mM dNTPs, Q5 hotstart polymerase, both Forward and Reverse Primers and autoclaved nanopure water.
Figure 2. 1kb DNA Ladder visualized by ethidium bromide staining on a 0.8% TAE agarose gel. Mass values are fore 0.5 µg/lane. Image of the ladder and the description was obtained from New England BioLabs Inc. website.
Analysis: Since the solution was approximately 220 µL which was oversized to run the PCR, solution was divided into four PCR tubes. Therefore, all four bands appear at the same location with the same intensity. Ironically, even though the secondary PCR did not clearly show the result in previous step, PCR^2 successfully worked by showing all four bands located at approximately 700~800 bases which was close to the actual DNA size, 731 nucleotides. From the result obtained from PCR^2, I could conclude that the gel was heated too long which probably made all samples to ran off.
LB agar plate or LB media - 09/26/13 3:48 ~ 07:26 PM
Objective: The purpose of this lab is to make 160mL of LB media and four agar plates using LB and Kanamysin for future experiment.
Analysis: Four LB agar plates were made using 80mL of LB and 80µL of Kanamycin. Kanamycin stock was used instead of Ampicillin stock, since pNIC-Bsa4 is Kanamycin resistance bacteria. On the other hand, antibiotic was not added into LB media.
PyMol Refresher - 09/29/13 3:30 ~ 6:45 PM
Objective: The purpose of this lab is to examine three dimensional structure of different enzymes.
Analysis: By comparing polar contacts, shape of inhibitor residues and binding mode at the active site, one could use these factors to determine similarities/differences of different enzymes. Moreover, one could figure out that each NAP residues are located at each chain from 3HBB enzymes, since the3HBB enzyme was composed of four different chains. Human and bacterial proteins were compared by aligning two proteins together using PyMol. By using BLAST, similarities/differences were determined.Different enzymes including 3HBB, 1U72, 3CL9 and 2H2Q are analyzed to find substrates, inhibitors and cofactors of different enzymes. Inhibitor MTX for human protein and TMQ for bacterial protein were similar, however, MTX binds better to the active site and has more residues to interact with.
PCR CleanUp & Nanodrop 10/01/13 2:18 ~ 3:12 PM
Objective: The purpose of this lab is to determine concentration of DNA by measuring absorbance by using spectrophotometry.
Figure 1. The concentration of cleaned PCR^2 product is measured via spectrophotometry. The concentration was 3.5 ng/µL. 260/280 was 1.81. 260/230 was 0.93.
Analysis: The absorbances at two different wavelengths were taken to estimate the purity of DNA eluted from PCR^2 samples. The concentration was 3.6 ng/µL which was incorrect, since the value for concentration was too low. Also, 260/280 was 1.81 which shows that the DNA sample used in this lab was pure since the ideal 260/280 is 1.8. 260/230 value - which shows the presence of other contaminants - was 0.93 which represents that the DNA sample was contaminated. DNA samples could possibly ran off with wash solutions which caused wrong concentration.
Transformation of competent cells for plasmid prep of pNIC-Bsa4 10/01/13 ~ 10/03/13
Objective: The purpose of this lab is to transform bacteria using E.coli DH5α and pNIC-Bsa4 to make more plasmid DNA.
Day 1 : 10/01/13 03:12 PM ~ 05:05 PM
Day 2: 10/02/13 6:00 ~ 6:34 PM
Day 3: 10/03/13 11:22 ~ 12:09 PM
Figure1. (From left to right) pallet obtained from the solution containing E.coli DHα5 (10µL) + pNIC-Bsa4 + LB + Kanamycin,
obtained from the solution containing E.coli DHα5 (50µL) + pNIC-Bsa4 + LB + Kanamysin, obtained from the solution containing E.coli DHα5 (50µL) + pNIC-Bsa4 + LB + Kanamycin, obtained from the solution containing E.coli DHα5 (10µL) + pNIC-Bsa4 + LB + Kanamycin
Analysis: On the first day, bacteria was incubated in LB agar plate with Kanamycin for approximately 25 hours. The next day, one bacteria colony was collected to place into LB media and Kanamycin was added which was finally placed in 37 degree shaker at fast rpm 250 rpm for 16 hours. On the last day, four flasks containing bacteria + LB media + Kanamycin was span down in 4 degree centrifuge at 6000 x g for 15 mins, and then only bacteria pallet was kept in -20 degree freezer.
Week 3 & 4
Kelly - good work. Include a ladder image. Dr. B 092713
PCR Primer Design Tails for pNIC-Bsa4 Cloning 09/12/2013 4:56 ~ 6:24 PM
Objective: The purpose of this lab was to design both Forward and Reverse primers for PCR amplifying the CDS of Alpha Carbonic Anhydrase and synthesizing ends of gene for the insertion into the pNIC-Bsa4.
Forward Primer
5' TAC TTC CAA TCC ATG AAA AAA ACC TTC CTG 3'
30 bp
GC Content: 36.7 %
Melt Temp
0mM Mg 2+: 58.1 C
1.5mM Mg 2+: 65.8 C
2mM Mg 2+: 66.4 C
4mM Mg 2+: 67.5 C
6mM Mg 2+: 68.1 C
Reverse Primer
5' TCT GCG AAA ACC CGT TAA CAG TAA AGG TGG ATA 3'
30 bp
GC Content: 42.4 %
Melt Temp
0mM Mg 2+: 62.6 C
0.5mM Mg 2+: 70.0 C
2mM Mg 2+: 70.5 C
4mM Mg 2+: 71.5 C
6mM Mg 2+: 72.0 C
Virtual Plasmid
NEB Cutter - Gel view
Figure 1. Gel view of pNIC-Bsa4 cut with Bsa I
Gel view of DNA sequence inserted in pNIC-Bsa4 cut with Bsa I was not found.
Analysis: LIC Cloning was done using the coding sequence for Alpha Carbonate Dehydratase with both upstream and downstream primers. The sequence was inserted to pNIC-Bsa4 and was cut with the restriction enzyme BsaI. However, get view of DNA sequence inserted into pNIC-Bsa4 which was cut with BsaI was not found.
Re-do PCR 09/13/2013 2:45 ~ 8:00 PM
objective: The purpose of this lab is to amplify the coding sequence in the pGBR-22 using Forward and Reverse Primers and using four different samples with different concentration of DNA plasmid.
Figrue 1. Picture of gel electrophoresis obtained from PCR trial 2 (re-done). Lane 1 shows 100bp marker (DNA Ladder). Lane 2 shows sample A with the concentration of 0.3 ng/µL pGBR-22 which obtained from 1:1000 template dilution. Lane 3 shows sample B with the concentration of 3 ng/µL pGBR-22 which obtained from 1:1000 template dilution. Lane 4 shows sample C with the concentration of 30 ng/µL pGBR-22 which obtained from 1:100 template dilution. Lane 5 shows the sample D with no plasmid included.
Figure 2. 100 bp DNA Ladder visualized by ethidium bromide staining on a 1.3% TAE agarose gel. Mass values are fore 0.5 µg/lane. Image of the ladder and the description was obtained from New England BioLabs Inc. website.
Analysis: pGBR-22 plasmid was diluted to either 1:1000 or 1:100 and the diluted plasmid was sued to create four different samples with different amount of plasmid. Lane 5, with no plasmid, did not have a band shown, since the sample did not have any DNA plasmid included. On the other hand, all other lanes clearly showed bands with different intensity. lane 4 had the highest intensity, meaning sample C had the most amount of DNA plasmid. However, it was hard to distinguish the different between land 2 and lane 3 due to the similar intensity which might have been resulted from dilution error. Moreover, the size of sample was approximately 1000 base pairs.
Further Analysis: According to NEBcutter, two cuts were approximately at 1000bp. These two cuts were similar to the lane 5 from figure 1 which indicates the site cut with PvuII enzyme.
Restriction Enzyme Digest 09/13/2013 1:25 ~ 8:00 PM
Objective: The purpose of this lab was to digest pGBR-22 plasmid with different restriction enzymes including EcoRI, PvuII and both EcoRI + PvuII and visualize fragments using gel electrophoresis.
Figure 1. Picture of gel electrophoresis obtained from Restriction Enzyme Digest. Lane 1 contains 1kb DNA Ladder. Lane 2 contains uncut pGBR-22 plasmid with the concentration of 48.95 ng/µL. Lane 3 contains pGBR-22 digested with BsaI. Lane 4 contains pGBR-22 digested with PvuII. Lane 5 contains pGBR-22 digested with both BsaI and PvuII.
Figure 2. 1kb DNA Ladder visualized by ethidium bromide staining on a 0.8% TAE agarose gel. Mass values are fore 0.5 µg/lane. Image of the ladder and the description was obtained from New England BioLabs Inc. website.
Analysis:
pGBR-22 plasmid was digested with BsaI instead of EcoRI. Wrong enzyme still cut the plasmid. Lane 3 showed that plasmid digested with BsaI was at 4kb. Lane 4 showed that plasmid digested with PvuII was at 3kb and 1kb. Lane 5 showed plasmid digested with PvuII and BsaI was at 1.5kb and 1kb. Lane 5 only showed two abnds which indicates that incorrect enzyme might cut the same site where PvuII enzyme would cut.
PCR Primer Overlap
Primer Dilutions for Assembly Step 09/12/2013 6:00 ~ 7:20 PM
Objective: The purpose for this lab was to make an oligo Mix consisting of all of the primers for the Primary PCR, Secondary PCR and the PCR^2.
Analysis: Oligo Mix was made for further experiment with the Primers obtained from IDT.
Analysis: Forward and Reverse primers for HpAlphaCA sequence were ordered and will be used for Secondary PCR.
I. Primary PCR 9/19/2013 3:00 ~ 6:30 PM
Figure 1. Picture of gel electrophoresis obtained from the primary PCR. Lane 1 is empty. Lane 2 contains 100 bp DNA ladder. Lane 3 contains primary PCR solution with oligo mix, dNTP, 5X rxn buffer, Q5 Polymerase and dH2O.
Figure 2. 100 bp DNA Ladder visualized by ethidium bromide staining on a 1.3% TAE agarose gel. Mass values are fore 0.5 µg/lane. Image of the ladder and the description was obtained from New England BioLabs Inc. website.
Analysis: Since oligo mix was added to the primary PCR solution, land 3 has a smear. However, even though a clear band did not occur, one could see a DNA was mostly gathered at 500 bp.
Primer Dilution 9/19/2013 4:00 ~4:30 PM
Objective: The purpose of this lab is to dilute primers ordered for cloning
Analysis: Each of HpAlphaCA forward and reverse primers was separately mixed with TE in order to make 100 µM of the stock solution to the original container. After the stock solution was made using TE and primer powder, working dilutions were made by adding 40 µL of forward and reverse primers separately into two different 1.7ml centrifuge tubes and adding 160 µL of autoclaved nanopure water into each of two tubes. Both stock solutions and working dilutions were stored in -20 C freezer for future experiment.
Week 1 & 2
PCR Primer Design for Primer Overlap Assembly PCR
Objective: The purpose of this lab is to design an Oligo primer set of forward and reverse for PCR using the gene obtained from pGBR-22.
Analysis: In this experiment, Oligo-primer for PCR for cloning was designed. Also, the gene sequence from NCBI was obtained from NCBI and the primer plate result from Helix system-CIT.
Quantifying DNA Using Nanodrop
Objective: The purpose of this lab is to measure the absorbance including 26/280 and 260/230 values and the concentration of pGBR-22 plasmid.
Figure 2. First result for pGBR-22 plasmid with the concentration of 162.2 ng/µL and the absorbance of 1.151.
Figure 3. Second result for pGBR-22 plasmid with the concentration of 164.8 ng/µL and the absorbance of 1.179.
Analysis: The graph shows that results obtained from Nanodrop was close to actual, since the actual concentration was 156.92 ng/µL and the experimental concentration was approximately 163.5 ng/µL. Also, we could conclude that there was very little contamination, since 260/280 and 260/230 were close to 1.8 and 2.1. Possible errors were contamination in samples and measurement errors.
Submitting DNA and DNA Sequencing Facility
Objective: The purpose for this lab is to add a primer to pGBR-22.
Analysis:
Analyzing DNA Sequence
Objective: The purpose for this lab is to determine the DNA sequence of pGBR-22 plasmid.
Analysis: DNA sequence of a plasmid was obtained from BLAST and sequence manipulation suite to get an entire sequence for pGBR-22 protein.
PCR